The osmolality of the blood in the renal inner medulla is high and varies with the urinary concentration. Both NaCl and urea are elevated. The medullary cells evidently survive and function in this adverse environment. The ongoing studies are concerned with understanding the mechanisms involved. When cells are stressed by a high salt environment, they generally accumulate osmotically active organic solutes ("osmolytes") in order to maintain a favorable internal milieu, while regulating their volume. We identified the organic osmolytes in renal inner medullary cells as glycerophosphocholine (GPC), betaine, sorbitol, taurine, and inositol, and showed that the osmolyte levels varied with urine concentration (and, presumably, medullary salt and urea concentrations). We are now using renal cell cultures and living animals to study the mechanism and control of osmoregulatory accumulation of these organic osmolytes, including the transcription factor (TonEBP/OREBP) that is involved. High NaCl signals activation of TonEBP via PKA, ATM, PARP-1, HSP90, PI3K and other systems. In addition, the acute response to high osmolality may include cell cycle arrest and apoptosis in proliferating cultures of renal medullary cells. High NaCl causes DNA damage in cell culture, while high urea causes both DNA (8-oxoguanine lesions) and protein(cabonylation) damage. Both the DNA damage and protein carbonylation are present in normal renal inner medullas. The DNA damage is rapidly repaired in vivo when renal inner medullary osmolality is reduced by the diuretic, furosemide. We are studying the mechanisms involved, including the role of p53, p38, Ku86, and GADD proteins.